LDRT combined with anti-PD-1 promotes tumor regression in head and neck squamous cell carcinoma through tumor metaprogram evolution and immune cell reprogramming.

BACKGROUND

Anti–PD-1 (αPD-1) therapy shows limited efficacy in head and neck squamous cell carcinoma (HNSCC), partly due to an immunosuppressive tumor microenvironment (TME) and insufficient infiltration of effector T cells. Low-dose radiotherapy (LDRT) has been suggested to modify the TME, potentially enhancing the effects of αPD-1.

METHODS

We profiled murine HNSCC treated with LDRT (1 Gy) and/or αPD-1 using single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq), complemented by orthogonal functional assays and in situ validation (spatial transcriptomics and multiplex immunofluorescence).

RESULTS

LDRT and αPD-1 synergistically suppressed tumor growth and remodeled the TME. Single-cell analyses showed malignant programs shifting from proliferative states toward stress- and interferon (IFN)-responsive states. Integrated scTCR-seq analysis revealed enhanced clonal expansion of tumor-reactive CD8⁺ T-cell subsets and reduced the frequency of CD4⁺ Treg-Ctla4 under combination therapy. TCR-based metrics (including STARTRAC) indicated higher migration potential and limited intratumoral clonal overlap for CD4⁺ Treg-Ctla4. An Isg15high macrophage state (Macro-Isg15) was positively associated with CD4⁺ Treg-Ctla4 frequency and was more prominent under αPD-1 monotherapy. Ligand–receptor inference highlighted a Macro-Isg15–associated CXCL14–CXCR4 chemokine axis enriched under αPD-1 monotherapy and attenuated in LDRT-containing regimens, suggesting reduced chemokine-driven support for CD4⁺ Treg-Ctla4 accumulation/recruitment.

CONCLUSION

These findings support an integrated framework in which LDRT augments αPD-1 efficacy by reprogramming malignant states and reshaping immune cell dynamics, including attenuation of immunosuppressive features within the TME. This preclinical evidence provides a rationale for translational evaluation of LDRT-based combination strategies in HNSCC.